Pre-mRNA splicing. The process of removing non-coding sequences (introns) from the transcripts of genes, is essential for viability in higher Eukaryotes. The splicing process involves hundreds of proteins and multiple RNA species which must come together in a precise sequence at the correct times, in order to remove the introns and nothing else. The multiple rearrangements and conformational changes that occur during splicing are hypothesized to be mediated by, among other things, RNA helicases, a class of proteins that unwind specific regions of RNA. The goal of this proposal is to investigate the role of a specific splicing factor, Brr2, in the splicing process and in the rearrangements that occur during splicing. One mutation in Brr2 blocks the first step in pre- mRNA splicing, and sequence similarity suggests that it is an RNA helicase. Furthermore, there is evidence that it interacts directly with the U2 snRNP. Implying that the latter may be its substrate. The experiments proposed here will identify the substrate of Brr2. look for its regulators, and define the function of a putative dsRNA-binding domain in Brr2. This work will extend our biochemical understanding of this crucial class of proteins and provide a foundation for future structural studies of helicases.